tbuckley/cabot-search huggingface dataset:
MDB-04. SINGLE-CELL TRANSCRIPTOMIC ANALYSIS REVEALS DISTINCT GENOMIC LANDSCAPES, CELL TRAJECTORIES, AND TUMOUR-MICROENVIRONMENT INTERACTIONS IN FOUR SUBTYPES OF MEDULLOBLASTOMA
Authors: Yana Ruchiy, Ioanna Tsea, Bronte Manouk Verhoeven, Indranil Sinha, Efthalia Preka, Thale Kristin Olsen, Shenglin Mei, Klas Blomgren, John Inge Johnsen, Lena-Maria Carlson, Cecilia Dyberg, Ninib Baryawno
Journal: Neuro-Oncology (2023)

Abstract Medulloblastoma is the most common paediatric high-grade brain tumour, which comprises four different molecular subtypes (SHH, Wnt, Group 3, and Group 4), each defined by different molecular characteristics, genomic aberrations, and prognosis. High levels of inter- and intra-tumoural heterogeneity, together with the detrimental side effects caused by the current medulloblastoma treatments, need to be addressed in order to develop more personalised treatments and improve the quality of life of these patients. In this study, we analysed single-cell RNA sequencing data from 14 medulloblastoma patients, comprising all four molecular subtypes. We found that these tumours exhibit great levels of heterogeneity with regard to their copy number variation (CNV) profiles analysed using Numbat, as well as immune cell infiltration and composition within these tumours. Tumour-infiltrating immune cells represented a heterogeneous population comprised of monocytes, M1- and M2-like macrophages, dendritic cells, and microglia. Our ligand-receptor analysis also identified a subset of immune cells that send EGF signals to the tumour clusters, potentially contributing to the tumour growth. In addition, we showed that the tissue of origin of the immune cells found within the tumour microenvironment and their medulloblastoma subgroup specificity greatly influence their clustering when projected onto a UMAP. We also found that the clustering of tumour cells is influenced by their CNV levels. Finally, we performed RNA velocity analysis and identified subgroup-specific cell trajectories for each medulloblastoma subtype. As predicted, identified cell trajectories demonstrated that cells move in a direction of increasing CNV levels, making it possible to visualise and track the tumour progression and identify genetic markers specific to either earlier or later tumour stages. Overall, our study underlines high levels of inter-tumoural heterogeneity in medulloblastoma. It also provides opportunities for the identification of subgroup-specific biomarkers for the early stages of medulloblastoma, potentially contributing to the discovery of novel drug targets.
BIOL-15. PATIENT-DERIVED XENOGRAFTS OF MATCHED PRIMARY-RECURRENT PEDIATRIC BRAIN TUMORS AS A RESOURCE FOR IDENTIFYING THERAPY RESISTANCE PROFILES
Authors: Miao Zhao, Thale Olsen, Tobias Bergström, Gabriela Rosén, Erika Dalmo, Elin Vikström, Ida Larsson, Tina Lin, Anders Sundström, Karl Holmberg, Holger Weishaupt, Sven Nelander, Geraldine Giraud, Fredrik Swartling
Journal: Neuro-Oncology (2023)

Abstract Tumor recurrence developing from therapy resistance, immune escape and metastasis is the leading cause of death of malignant pediatric brain tumors. By studying paired primary-recurrent patient samples we previously identified significant accumulation of SOX9-positive cells in Group 3 medulloblastoma that was similarly induced following long-term irradiation in transgenic mouse models of MYC-induced medulloblastoma. To follow relapse biology in malignant pediatric brain tumors at the single cell level we have established patient-derived xenografts (PDXs) from a number of matched primary-relapse samples, including pediatric high-grade glioma, medulloblastoma and ependymoma profiled with DNA methylation analysis. We next used scRNA-Seq to identify clones with particular expression of genes and distinct signaling pathways enriched in recurrent tumor cells as compared to their matched primary tumor cells. We further used a lentivirus-based barcoding strategy coupled to scRNA-Seq to study the molecular evolution of cell clones arising from genetically modified mouse models and PDXs that survive long-term fractionated irradiation, cisplatin or temozolomide treatment in vitro and in vivo. By using computational modeling we identified means of specific targeting of recurrent cells that could be of potential use in future treatments for patients affected by fatal relapses. Our results suggest that pediatric brain tumors of distinct tumor entities use different ways to acquire therapy resistance in patients which is often coupled to an expansion of clones that are developing therapy-induced resistance.
EPEN-10. SPINAL EPENDYMOMA WITH MYCN-AMPLIFICATION – A DISEASE OF CHILDHOOD AND YOUNG ADULTHOOD WITH DISMAL PROGNOSIS
Authors: David R Ghasemi, Martin Sill, Konstantin Okonechnikov, Margaret Shatara, Stephen Yip, Zita Reisz, Karl-Heinz Plate, David Scheie, Benedikte Hasselbalch, Hildegard Dohmen, Amel Chaouch, Nada Jabado, Wilfred F A den Dunnen, Pieter Wesseling, Stefan Fröhling, Andrew Donson, David Capper, Ulrich Schüller, Stefan M Pfister, David E Reuss, Kristian W Pajtler
Journal: Neuro-Oncology (2023)

Abstract Spinal ependymoma (EPN) with MYCN-amplification (SP-EPN-MYCN) was recently recognized as a distinct tumor type within the 2021 WHO classification of CNS tumors. To date, information about the epidemiological, clinical, and biological features of SP-EPN-MYCN remain sparse. Using DNA-methylation profiling, we collected a cohort of n = 71 (54 primaries, 17 relapses) SP-EPN-MYCNs. Based on the Heidelberg Methylation Brain Tumor Classifier V12.5, 95.6% (68/71) reached a classifier score of ≥ 0.9. In 68/71 cases, focal amplifications of MYCN were detected by copy number variation (CNV) analysis. Two tumors of pediatric patients harbored MYC-amplifications. Other repeated CNVs amongst the primary cases included gains of chromosomes 5 (5/54) and 18 (5/54), loss of chromosome 10 (14/51), and combined 17p loss and 17q gain (5/54). FISH (n = 17) and immunohistochemistry (n = 19) provided positive results for the detection of the MYCN-amplification. Epidemiological data was available for 63% (34/54) of all patients, showing an even sex distribution (17 female patients). Twenty-four percent (8/34) were children and adolescents younger than 20 years. The rest of the cohort consisted of an AYA-population with an overall median age of 31.5 years. Primary lesions were predominantly located in the thoracic (72%) and/or cervical (55%) spinal cord. Two patients showed extra-CNS metastasis to the humerus and the paraspinal musculature, respectively. At the point of analysis, 38% (10/26) of all patients had died, and 10/16 alive patients had already developed disseminated disease. Of patients with available information, 90% (18/20) had relapsed, whereas 10% (2/20) showed stable disease with leptomeningeal spread. Median PFS (n = 17) and OS (n = 25) were 15,5 (12 – 34) and 85 (61 – NA) months. In summary, SP-EPN-MYCN is a disease with dismal prognosis that can disseminate outside of the CNS and affects mainly children and AYA. Currently, comprehensive multi-omics analyses are ongoing.
IMG-02. VISUALIZATION OF TIGIT IN THE GLIOMA MICROENVIRONMENT: THE CREATION OF A PET IMAGING TRACER
Authors: Stephen Frederico, Sarah Vincze, Michal Nisnboym, Bo Li, Chaim Sneiderman, ReidAnn Server, Kathryn Day, Joseph LaToche, Jessie Nedrow, Wilson Edwards, Gary Kohanbash
Journal: Neuro-Oncology (2023)

Abstract Pediatric high-grade gliomas (HGGs) are aggressive tumors and are one of the drivers of pediatric brain tumor mortality. Few effective treatment options exist for pediatric HGGs beyond surgery and chemoradiation. Additionally, some HGGs, such as diffuse midline glioma, are often surgically inaccessible and respond poorly to chemotherapy leaving radiotherapy as the only treatment for this disease. While immune checkpoint inhibitors (ICIs) have seen success in treating cancers outside the CNS, ICIs for gliomas have shown limited efficacy. TIGIT is an IC receptor that is expressed on activated T-cells and has a critical role in suppressing T-cell and NK cell function. Prior work by our group has shown TIGIT to be a promising target for adult gliomas. These studies included analysis of transcriptomic data, and preclinical studies in glioma-bearing mice. We therefore assessed pediatric glioma transcriptomic datasets and identified via optimal cutoff analysis that at a specific mRNA threshold, TIGIT expression is associated with worsened survival. As TIGIT expression may be both prognostic and a biomarker for anti-TIGIT ICI therapy, we created a radiolabeled immunoPET tracer capable of binding TIGIT antigen that could be visualized via PET imaging. Referred to as “89Zr-DFO-TIGIT”, we found this tracer bound TIGIT with moderate immunoreactivity (57.7% ± 3.5%) as determined by bead-based immunoreactivity assays. As proof of feasibility, we injected glioma-bearing mice (GL261 cell line) with 89Zr-DFO-TIGIT which enabled the visualization of TIGIT in the tumor microenvironment via PET imaging. Thereafter, we performed biodistribution studies which found there to be significantly increased 89Zr-DFO-TIGIT in mouse tumor-bearing hemispheres as compared to non-tumor-bearing hemispheres. PET imaging also showed tracer-to-tumor uptake increased from day one to day eight. Our findings demonstrate the capability of 89Zr-DFO-TIGIT to visualize and quantify TIGIT in the glioma microenvironment, highlighting how this tracer may be of value in pediatric brain tumor immunotherapy studies.
METB-02. DECIPHERING THE CELLULAR HETEROGENEITY OF INFANT-TYPE HEMISPHERIC GLIOMAS USING SINGLE-NUCLEI MULTI-OMICS
Authors: Andrea J De Micheli, Michal Zápotocký, Florence M G Cavalli, Ana S Guerreiro Stücklin
Journal: Neuro-Oncology (2023)

Abstract Infant-type hemispheric gliomas (IHGs) have recently been recognized as a new tumor entity. Compared to other types of pediatric gliomas, most IHGs distinctively harbor a fusion in a cluster of receptor tyrosine kinase (RTK) genes: the NTRK family, ROS1, ALK, or MET. Clinically, IHG patients appear to be associated with better outcomes compared to older children with high-grade tumors. While large-scale genomic studies have helped uncovered the complex molecular landscape and clinical features of IHGs, little is known about the underlying biology – specifically how tumor heterogeneity may explain the spectrum of clinical presentations and progression of the disease. Using single-nuclei multi-omic (RNA/ATAC-seq) profiling, we assembled a comprehensive atlas of IHG tumor cells from a cohort of patients spanning different age groups and RTK fusion subtypes. We measured that tumor single-nuclei transcriptomes clustered into distinct subpopulations and contained genes expressed by the normal brain cell lineages. Data integration with single-cell atlases from the developing brain allowed us to rank tumor cell subpopulations according to their developmental stage, with age- and fusion-specific differences. Using trajectory inference and gene expression velocity modeling, we describe a hierarchical and dynamic organization between subpopulations, potentially illustrating the role of phenotypic plasticity in glioma formation. Moreover, we measured in fragments of open chromatin a cell-type specific enrichment of binding sites for transcription factors known to regulate brain cell maturation. Taken together, our multi-modal atlas describes the cellular and functional heterogeneity of IHGs, paving the way towards a greater understating of its unique biology and clinical consequences.
EPEN-09. UTILITY OF RADIATION THERAPY IN SPINAL CORD MYXOPAPILLARY EPENDYMOMAS: A REVIEW OF THE LITERATURE
Authors: Kasey Jackson, Sarah Kleinknecht, Roger Packer, Eugene Hwang
Journal: Neuro-Oncology (2023)

Abstract INTRODUCTION Myxopapillary ependymomas (MPEs) are slow-growing tumors that comprise about 25% of spinal ependymomas. Although historically designated as grade 1, the recent WHO classification has upgraded MPEs to grade 2 given their potential for recurrence and dissemination. While the National Comprehensive Cancer Network guidelines recommend avoiding radiation therapy (RT) unless a gross-total resection (GTR) is not achieved, several reports have suggested a potential benefit of adjuvant RT in improving relapse-free survival in children after GTR. This literature review was conducted to compare available data to determine the utility of upfront irradiation in these patients. METHODS A comprehensive literature review from 1984-2021 was performed on PubMed using keywords: “pediatric myxopapillary ependymoma” and “radiation.” One case was censored for unknown radiation status. RESULTS 17 analyses from 1984-2021 were reviewed, yielding 140 pediatric cases of MPE, of whom 59% (n=83) received a GTR. Of those that received a GTR, average age was 13.7 yrs (range, 6-21), and 66% were male. The most common location was lumbosacral (25%), followed by thoracolumbar (19%). 22.8% (n=19) of patients received upfront focal RT. Relapse occurred in eight (42%) patients who received upfront RT, and 30 (47%) of those who did not, with no clear age or gender discrimination in those that relapsed. Distant recurrence occurred in 75% of those who received RT, and 68% of those that did not. The duration of event-free survival will be included in the final presentation. CONCLUSIONS While some individual studies have suggested a survival advantage in pediatric MPE after focal RT, in this meta-analysis, both relapse rate and location seemed similar regardless of receipt of RT. From this available evidence, the efficacy of upfront RT has not been demonstrated.
BIOL-12. ENHANCING IMMUNE-MEDIATED TUMOR CONTROL IN MURINE MODELS OF GROUP 3 MEDULLOBLASTOMA
Authors: Zahra Abbas, Hilary Hii, Jessica Buck, Iley Johnson, Jacob Byrne, Omar Elaskalani, Mani Kuchibhotla, Courtney George, Clara Andradas Arias, Meegan Howlett, Joost Lesterhuis, Terrance Johns, Nicholas Gottardo, Raelene Endersby
Journal: Neuro-Oncology (2023)

Abstract Stagnation in clinical outcomes as well as severe side-effects of current treatment approaches for Group 3 medulloblastoma have prompted the search for novel therapies. To date, immunotherapies have not demonstrated success in medulloblastoma. Due to the highly aggressive nature of relapsed medulloblastoma, we suggest that it would be advantageous to introduce new immunotherapies first-line, alongside conventional therapies. To design immunotherapeutic strategies that are compatible in combination with standard therapies, we first determined the effect of standard-of-care craniospinal irradiation (CSI) and chemotherapy on the immunology of these tumours in immune competent and deficient murine models. We show that standard treatment approaches deplete adaptive immune cells, while macrophage and brain-resident microglia are not depleted. Furthermore, medulloblastomas are myeloid dominant tumors, and blocking the anti-phagocytic CD47-SIRPa interaction has shown preclinical efficacy. Therefore, we tested the combination of anti-CD47 immunotherapy with radiotherapy in a patient-derived xenograft model of Group 3 medulloblastoma. We found that anti-CD47 monotherapy had limited efficacy, while CSI results in only a temporary reduction in tumor size. In the cerebellum of relapsed tumors post-CSI, flow cytometry revealed increased peripheral myeloid populations. Remarkably, the combination of anti-CD47 and CSI was effective in clearing tumors and maintaining long-term tumor-free survival. Using RNA sequencing and immunohistochemistry, we found that the combination therapy results in enhanced cytokine signalling and phagocytic myeloid cell infiltration into the tumors compared to the monotherapies. We have also developed and characterized novel age-appropriate preclinical models for medulloblastoma and have observed altered myeloid cell phenotypes in these age-appropriate models. We are now testing this novel combination therapy in our new neonatal murine models to determine efficacy in age-appropriate hosts.
MDB-33. REST AND USP37 CONTROL THE MTORC1-AUTOPHAGY AXIS IN MEDULLOBLASTOMA BY REGULATING RAPTOR DEGRADATION
Authors: Donghang Cheng, Ashutosh Singh, Amanda R Haltom, Yan Zheng, Ajay Sharma, Mariana Lopez, Nancy Gordon, Vidya Gopalakrishnan
Journal: Neuro-Oncology (2023)

Abstract The RE1 Silencing Transcription Factor (REST) is a repressor of neurogenesis. Its expression is elevated in the pediatric brain tumor medulloblastoma (MB) and correlates with poor patient prognosis. RNAseq analyses of MB tumors identified significant differences in the expression of genes associated with autophagy between high- and low-REST expressing samples. Microscopy and biochemical analyses of isogenic high- and low-REST human MB cell lines confirmed REST-dependent changes in autophagy, suggesting that REST controls the process. We had previously shown that REST regulates the activity of the mammalian target of rapamycin complex 2 (mTORC2). However, whether REST controls the mTORC1 pathway, a negative regulator for autophagy, is still unknown. mTORC1 is known to block autophagy initiation by phosphorylation of UNC51-like autophagy activating kinases (ULK)1/2 and the Phosphatidyl Kinase 3 subunit C3 (PIK3C3) to prevent global expression of lysosomal and autophagy genes through Transcription Factor EB (TFEB) phosphorylation. The above findings led us to investigate if REST controls autophagy through modulation of mTORC1 activity in MBs and to understand the underlying mechanisms. Biochemical studies demonstrated that REST elevation promotes proteasomal degradation of Raptor, a key regulatory sub-unit of the mTOR1 complex, to induce autophagy. Specifically, we discovered that Raptor is a novel substrate of the deubiquitylase USP37, which we previously identified as a REST-target gene product. These data show that REST drives autophagy by silencing USP37, destabilizing Raptor, and blocking mTORC1 activity. Finally, the pharmacological blockade of autophagy caused a decline in MB cell growth in a REST-dependent manner, indicating that REST-dependent autophagy is a pro-survival mechanism in MBs.
OTHR-08. MULTIDISCIPLINARY APPROACH TO PAEDIATRIC BRAIN CANCER BIO-BANKING IMPROVES ENROLLMENT, FACILITATES DISCOVERY RESEARCH, AND ENABLES BETTER ACCESS TO DIAGNOSTIC AND THERAPEUTIC STUDIES
Authors: Nataliya Zhukova, Dilru Habarakada, Caroline Drinkwater, Andrew Danks, Chris Xenos, Beena Kumar, Lauren Haddad, Paul Daniel, Claire Sun, Paul Wood, Leanne Super, Lisa Janson, Jason Cain, Ron Firestein, Peter Downie
Journal: Neuro-Oncology (2023)

Abstract Despite the significant progress made in the multi-modal treatment of childhood malignancies over the last four decades with a concomitantly increased cure rates, the benefit is still largely limited to patients with leukemias, lymphomas and localized non-CNS solid tumors, leaving patients with high-risk non-CNS solid tumours and most CNS malignancies with minimal advancements. More research is critical to understand what drives these cancers and to investigate the best ways to deliver curative therapies. Translational research relies on patient-derived tissue samples, animal models and cell-cultures to understand the biological, genetic, and molecular mechanisms of disease. To facilitate patient care and outcomes it is becoming increasingly important that paediatric clinical trials include tissue availability as part of eligibility criteria, making collection and storage of patient tissue a mandate of personalized medicine and a pillar of modern paediatric cancer medicine. Monash Children’s Cancer Biobank was established in 2011. Since 2017, a total of 64 patients were diagnosed with CNS malignancies across all grades, with an overall 69% of patients’ tissues being bio-banked at the time of initial diagnosis. Through the combination of an educational forum, regular multi-disciplinary meetings, and the early involvement of medical oncology and biobank staff, with our neurosurgical and clinical pathology colleagues, as part of patient management planning, the tissue acquisition for bio-banking increased from 44% to 82% over the course of 5 years. This consequently led to a 100% enrollment to tissue-enabling studies allowing for banking of both fresh-frozen tissue and living cell-line models, facilitating discovery research efforts as part of the Victorian Paediatric Cancer Consortium’s molecular analysis pipeline (https://vicpcc.org.au/dashboard). This significantly improves opportunities for identification of targetable pathways, access to targeted therapies, enrollment onto treatment clinical trials and identification of patients with cancer predisposition syndromes.
EPEN-04. DEVELOPMENT OF B7-H3-TARGETED CAR T CELL THERAPY FOR EPENDYMOMA
Authors: Sanya Mehta, Haley Houke, Jorge Ibanez, Erik Emanus, Stephen Mack, Giedre Krenciute
Journal: Neuro-Oncology (2023)

Abstract Targeted treatment options are desperately needed for ependymomas (EPN). Chimeric antigen receptor (CAR) T cells have immense potential to positively transform treatment outcomes; however, little is known regarding antitumor efficacy of CAR T cells against EPNs. We found that B7-H3 is highly expressed on pediatric brain tumor patient-derived orthotopic xenografts from EPN patients, making it a promising target for CAR-T cells. The project’s goal is to develop a safe, effective B7-H3 targeted CAR T cell immunotherapy for EPN. We generated second generation human B7-H3-CAR T cells and examined their anti-EPN cytolytic activity, expansion, and cytokine production. We found that B7-H3-CAR T cells had potent in vitro antitumor activity against all EPN cell lines tested (1425, ST1, CPITT, ST2, EPI, L6SJ). In repeated-stimulation assays, CAR T cells expanded 7-10 times, and up to one million-fold when co-cultured with EPN cells. Interestingly, while the EPN cells were found to secrete high levels of C-C motif chemokine ligand 2 (CCL2) which is consistent with published data, our Multiplex analysis of co-culture supernatants shows that CCL2 production increased up to 10-fold when EPN cells were co-cultured with CAR T cells. Finally, we demonstrate that B7-H3-CAR T cells had antitumor activity in a supratentorial-EPN xenograft mouse model (1425 cell line) without apparent toxicities, although 4 out of 12 tumors re-lapsed. Recurrent tumor analysis is underway. Together, our data shows establishment of a clinically relevant model to study resistance to CAR T cell therapy in EPN, and preliminary mechanistic insight into CAR T cell persistence and modulation through CCL2 signaling. Given that little is known about the function of CCL2 and its receptor, C-C chemokine receptor type 2 (CCR2) in EPNs or CAR T cells, future work will focus on defining these biological mechanisms, enabling us to develop improvements to B7-H3-CAR T cells for EPN.